Resultados y análisis de uso y cobertura del suelo

Feigner and Ringold observed that DNA and DOTMA containing liposomes, form complexes spontaneously within an aqueous environment/^ Their observations suggested that almost all the DNA of the complex was trapped in the interior of the complex^"^ and they hypothesised that the optimum DNA to cationic liposome ratio was when charge neutralisation had occurred. Furthermore, they also postulated that the cationic liposome / DNA complex was formed from one molecule of plasmid DNA and

four cationic liposomes (Figure 1.2.16).^^ This rationale was based upon the

assumption that the plasmid DNA contained 2500 base pairs and hence 5000 negative charges. The liposome was made up of DOTMA / DOPE (1:1) and the average diameter of the liposome was 250 nm, thus approximately 2500 lipid molecules and hence 1250 DOTMA molecules. For charge neutralisation to occur, four cationic liposomes would have to complex to one DNA plasmid.^^

Cationic Liposome

DNA Liposome / nucleic acid

Complex

Figure 1.2.16 The co-ordination of 4 cationic liposomes to plasmid DNA75

Unfortunately this model is flawed; it assumed that the entire cationic lipid is concentrated on the outer membrane of the liposome whereas in reality cationic lipids

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supercoiled, resulting in a change of dimensions for the complex and ultimately making the complexation o f four liposome molecules around the DNA plasmid untenable/^

Smith et a l suggested a series o f possible liposome/DNA structures based on

unilamellar l i p o s o m e s ( i ) The electrostatic model, (ii) The internal model, (iii) The coated electrostatic model, (iv) The mixed model.

(i) The electrostatic model.

This model is based upon a supercoiled DNA plasmid 70 nm in length and 5 nm in diameter and they proposed that several cationic liposomes, 250 nm in diameter, could aggregate around the plasmid. The number o f liposome molecules needed for

these interactions to occur was not indicated (Figure 1.2.17).

70nm

Plasmid DNA

Cationic Liposome

250 nm

Figure 1.2.17 The electrostatic model. The interaction of plasmid DNA to the surface of a liposome is shown.*’

(ii) The internal model.

The second aggregate considered was where the plasmid DNA was completely

internalised within a liposome (Figure 1.2.18).^^ This theory is an extension o f the

concept o f the internalisation of ionisable drugs. There are two possible scenarios:

(a) where the DNA binds the inner surface of the liposome and (b) where the DNA has no interactions with the liposomal membrane.

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A _B

Figure 1.2.18 The internal model. The encapsulation of plasmid DNA in a cationic liposome is shown,

A) where the DNA binds the inner surface and B) where it is non-bound.'^

Conversely, Sternberg et al investigated DNA/cationic liposome aggregates using freeze-fracture electron microscopy and suggested that the DNA aggregated to the outer membranes of the cationic liposomes/^

(iii) The coated electrostatic model.

The third model suggests that the DNA is coated in a lipid bilayer and the aggregation o f this complex would result in a multi layered DNA/lipid structure

(Figure 1.2.19).17 = L ip id M o lec u le =dna N e u tr a l L ip id L o c a lisa tio n üüuyuüüüuuuüuüüüuuüu

uuuuuuuüuuüuuuuyuyuü

yyyyyyywyyyyyyyyyyyy

Figure 1.2.19 The coated electrostatic model. A) shows DNA covered in cationic lipid bilayers. B)

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Kikuchi and Carmona-Ribeiro supported this theory with investigations into the

interactions between X bacteriophage double stranded DNA and a cationic liposome

containing dioctadecyldimethylammonium bromide (DODAB)/^ They envisaged the formation o f an intermediate, whereby each strand o f the DNA is covered in a DODAB bilayer. The resulting bilayered structure was found to be unstable and led to the dissociation o f one bilayer from the complex, resulting in the formation of a “globule”,

where the DNA was sandwiched between lipid monolayers (Figure 1.2.20)/^

= Lipid

= DNA

= W ater

2 .5 n m 1 .3 n m 1 n m

Figure 1.2.20 DNA/DODAB complexes.77

7 8

Investigations by Lasic et al. supported the formation o f these aggregates.

Cryo-electron microscopic investigations indicated that DNA was absorbed between cationic lipid bilayers as a single layer of parallel helices and resulted in the formation of stacked DNA / liposome structures.^^

(iv) The mixed model.

The fourth model suggests the DNA can exist both in the interior and exterior of liposomes whereby the DNA maximises its association with the liposome.

Sternberg et a l have investigated DNA/cationic liposome (DC Choi and DOPE)

complexes using freeze-fracture electron microscopy.^^ The aggregates formed, resembled liposome complexes covered in DNA tubules (“spaghetti and meatballs”)

(Figure 1.2.21). Sternberg et a l postulated that the DNA acted as a fusogenic agent, aggregating positively charged liposomes to form semi-fused complexes.^^ The tubules

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had diameters o f approximately 7 nm. Furthermore, they suggested that the spaghetti and meatball complexes would form under conditions for optimal transfection using DC

Chol/DOPE liposomes.76 Cationic Liposome DNA V A Liposome/DNA Aggregate Spaghetti/meatball complexes (Liposome/DNA assemblies)

Figure 1.2.21 Spaghetti and meatball complexes.

The complexes discussed may be too simplistic, for example, Gustafsson et a l

visualised various cationic liposome/DNA complexes using cryo-transmission electron microscopy.^^ Their results suggested that for lipid/DNA charge ratios greater than one, a complex is formed which leads to the entrapment of DNA molecules within multilamellar structures.^^ Furthermore, a significant quantity o f free plasmid DNA (uncomplexed) was observed in the region o f the aggregates.^^ The existence of isolated DNA molecules is not suitably represented by Smiths’ m o d e l s , a n d the observation o f formation of multilamellar structures indicates that cationic liposome/DNA interactions are far more complex. Multilamellar structures have been

detected by X-ray diffraction data obtained by Radier et The DNA was

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aggregates that have a reduced electrostatic repulsion to cationic liposomes. This allows for the formation of the multilamellar structures, driven by the release of DNA associated counter ions (Figure 1.2.22),^^ The multilamellar complexes were observed at the liposome/DNA charge neutralisation ratio, and since transfection was most efficient at this ratio,^^ suggested that these multilamellar structures may be involved the types o f DNA/liposome complexes that associate with membranes.

Figure 1.2.22 Representation of the arrangement of DNA and lipid bilayers. The DNA is represented

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